WO2014005298A1 - Kit elisa de détection lié à l'enzyme dinitolmide et son application - Google Patents

Kit elisa de détection lié à l'enzyme dinitolmide et son application Download PDF

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Publication number
WO2014005298A1
WO2014005298A1 PCT/CN2012/078164 CN2012078164W WO2014005298A1 WO 2014005298 A1 WO2014005298 A1 WO 2014005298A1 CN 2012078164 W CN2012078164 W CN 2012078164W WO 2014005298 A1 WO2014005298 A1 WO 2014005298A1
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WIPO (PCT)
Prior art keywords
dinitramine
enzyme
antibody
solution
hapten
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PCT/CN2012/078164
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English (en)
Chinese (zh)
Inventor
何方洋
冯才伟
万宇平
徐念琴
罗晓琴
冯静
陶光灿
余厚美
李勇
扶胜
吴鹏
崔海峰
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北京勤邦生物技术有限公司
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Priority to PCT/CN2012/078164 priority Critical patent/WO2014005298A1/fr
Priority to US14/412,317 priority patent/US10234470B2/en
Publication of WO2014005298A1 publication Critical patent/WO2014005298A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes

Definitions

  • Enzyme-linked immunosorbent kit for detecting dinitramine and application thereof
  • the present invention relates to an enzyme-linked immunoassay technique, and in particular to an enzyme-linked immunoassay kit for detecting dinitramine and an application thereof. Background technique
  • Dinitramine is a nifediamide anticoccidial drug developed by Dow Company of France in 1960. It was approved for production in China in 1989. Dinitramine is widely used because of its good effect on the control of coccidia, but in use, If the dosage is too large or continues to be used in laying hens and in the withdrawal period, it will cause residual drug residues in animal products, which may cause potential harm to human health.
  • the Ministry of Agriculture's Bulletin No. 235 stipulates the maximum residue limit of dinitramine, chicken. And chicken liver were 3000 g/kg and 6000 g/kg, respectively. There is currently no corresponding standard method for residual analytes, so it is necessary to carry out an analytical method for the determination of dinitramine residues.
  • the enzyme-linked immunosorbent kit for detecting dinitramine comprises: a dinitramine specific antibody, a coating original and an enzyme label; wherein preferably the dinitramine-specific antibody is dinitrazole An amine monoclonal antibody or a dinitramine polyclonal antibody, preferably the coating is a conjugate of a dinitramine hapten and a carrier protein, or a dinitramine-specific antibody or an anti-antibody, and preferably
  • the enzyme label is an enzyme-labeled goat anti-mouse anti-antibody or a goat anti-rabbit anti-antibody, an enzyme-labeled anti-dinitramine monoclonal or polyclonal antibody or an enzyme-labeled dinitramine hapten.
  • the enzyme label is an enzyme-labeled goat anti-mouse anti-antibody or a goat anti-rabbit anti-antibody or an enzyme-labeled anti-dinitroto An amine monoclonal or polyclonal antibody; when the coating is originally a dinitramine-specific antibody or an anti-antibody, the enzyme label is an enzyme-labeled dinitramine hapten.
  • the carrier protein used in the present invention is any protein capable of forming an immunogen after coupling with a dinitramine hapten, preferably thyroprotein, bovine serum albumin, murine serum albumin, rabbit serum albumin, human serum albumin, blood blue Protein, fibrinogen or ovalbumin.
  • the kit may also include dinitramine standard solution, color developing solution, concentrated washing solution, stop solution, and/or concentrated complex solution.
  • the concentrated washing liquid may be a pH 7.1-7.6, 0.2-0.4 mol/L phosphate buffer containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide;
  • the solution may be a pH 7.2-7.8, 0.1-0.2 mol/L phosphate buffer;
  • the coloring liquid is composed of a coloring liquid A liquid and a color developing liquid liquid B, the color developing liquid A liquid may be hydrogen peroxide or urea peroxide, and the color developing liquid liquid B may be o-phenylenediamine or tetramethyl combined.
  • Aniline; the stop solution may be a 0.5 to 2 mol/L sulfuric acid or hydrochloric acid solution.
  • Tween-20 in the buffer will reduce the non-specific adsorption of the antibody, and also protect the protein from azide.
  • sodium azide inhibits the growth of bacteria in solution and provides a protective effect on the stability of the solution.
  • the dinitramine hapten can be obtained by catalytic hydrogenation of dinitramine (eg Pd/C) to obtain a monoamino derivative, and then reacting with succinic anhydride to obtain a dinitramine hapten (5-amino nitrate). Tolamine monosuccinic acid amide).
  • the dinitramine specific antibody is obtained by using a conjugate of the dinitramine hapten and a carrier protein as an immunogen;
  • the carrier protein is thyroprotein, bovine serum albumin, murine serum albumin, rabbit serum Protein, human serum albumin, hemocyanin, fibrinogen or ovalbumin.
  • Dinitramine is a small molecule substance that is immunoreactive, non-immunogenic, and does not induce an immune response in the body. It must be coupled to a macromolecular carrier protein to be immunogenic.
  • the present invention couples the dinitramine hapten to the carrier protein by the carbodiimide method, highlights the characteristic structure of the dinitramine, and also increases the immunogenicity and specificity of the dinitramine hapten.
  • the dinitramine monoclonal antibody and/or polyclonal antibody may be a murine, equine, sheep, rabbit or guinea pig antibody.
  • the dinitramine monoclonal antibody is an antibody secreted by a monoclonal hybridoma cell line E-2-4 of dinitramine having a deposit number of CGMCC No. 6143.
  • the enzyme-labeled anti-antibody is obtained by coupling the labeling enzyme with the anti-antibody by a sodium periodate method; in the sodium periodate method, the molar concentration of the labeling enzyme and the anti-antibody Ratio is 2: 1 ;
  • the enzyme is alkaline phosphatase or horseradish peroxidase, preferably horseradish peroxidase.
  • the improved sodium periodate method omits the step of blocking the amino group on the enzyme, which saves time and reduces the ratio of horseradish peroxidase (HRP) to anti-antibody concentration, saving raw materials.
  • Another object of the present invention is to provide a method of detecting dinitramine.
  • the method for detecting dinitramine comprises the following steps:
  • sample preparation Weigh the sample into a centrifuge tube, extract with acetonitrile, centrifuge to precipitate, take the supernatant and dry it in a water bath, add n-hexane to dissolve impurities, extract with working complex solution, centrifuge Thereafter, the lower layer solution is taken for analysis, wherein the sample is an animal feed or animal tissue sample, preferably chicken feed, chicken or chicken liver;
  • the mouse monoclonal hybridoma cell line E-2-4 having the accession number CGMCC No. 6143 is also within the scope of the present invention.
  • the enzyme-linked immunosorbent kit for detecting dinitramine mainly uses a competitive ELISA method to qualitatively or quantitatively detect the residual amount of dinitramine in the sample; the kit has low requirements for pretreatment of the sample, and the sample preparation process is simple and capable. At the same time, rapid detection of large quantities of samples; the main reagents of this kit are provided in the form of working fluids, the test method is convenient and easy, and has the characteristics of high specificity, high sensitivity, high precision and high accuracy.
  • the enzyme-linked immunoassay kit of the invention has the advantages of simple structure, convenient use, low price, convenient carrying, high and accurate detection method, convenient and on-site monitoring, and suitable for qualitative and quantitative screening of a large number of samples, which will be used in dinitramine Play an important role in the detection.
  • Figure 1 is a schematic diagram showing the synthesis of dinitramine hapten
  • Fig. 2 is a standard curve of a kit in which a conjugate of a dinitramine hapten and a carrier protein is used as a coating. detailed description
  • the enzyme-linked immunosorbent kit for the coating of dinitramine hapten and carrier protein conjugates generally includes:
  • Enzyme-labeled anti-antibody working solution goat anti-mouse anti-antibody labeled with horseradish peroxidase or goat anti-rabbit anti-antibody labeled with horseradish peroxidase;
  • the substrate coloring solution is composed of liquid A and liquid B.
  • the substrate coloring liquid A is urea peroxide or hydrogen peroxide
  • the substrate coloring liquid B is tetramethylbenzidine or o-phenylenediamine.
  • the stop solution is 0.5-2 mol/L hydrochloric acid or sulfuric acid;
  • the concentrated washing solution is a 0.2-0.4 mol/L phosphate buffer solution having a pH of 7.1-7.6, containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide;
  • the concentrated solution is a phosphate buffer solution of pH 7.2-7.8 and 0.1-0.2 mol/L.
  • the enzyme-linked immunosorbent kit for the coating of the dinitramine hapten and carrier protein conjugate of the present invention comprises:
  • Enzyme-labeled anti-antibody working solution goat anti-mouse anti-antibody labeled with horseradish peroxidase;
  • the concentration is 0 g / L, 1 g / L, 3 g / L, 9 g / L, 27 g / L, 81 g / L;
  • the substrate color developing solution is composed of liquid A and liquid B, the substrate coloring liquid A liquid is urea peroxide, and the substrate color developing liquid liquid B is tetramethylbenzidine;
  • the stop solution is 0.5-2 mol/L sulfuric acid
  • the concentrated washing solution is a 0.2-0.4 mol/L phosphate buffer solution having a pH of 7.1-7.6, containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide.
  • the concentrated solution is a phosphate buffer solution having a pH of 7.2-7.8 and 0.1-0.2 mol/L.
  • the dinitramine hapten and bovine serum albumin (BSA) obtained above were subjected to the carbodiimide method (see Nanjing University Press, Yang Liguo, Hu Shaozhen, Wei Pinghua, etc., "Enzyme Immunoassay Technology” P254-255) Coupling was carried out to obtain an immunogen.
  • the dinitramine hapten and the ovalbumin (purchased from Shanghai Xibao Biotechnology Co., Ltd.) were coupled by a carbodiimide method to obtain a coating original.
  • the spleen cells were taken and fused with SP2/0 myeloma cells (purchased from the Institute of Genetics and Developmental Biology, China) in a ratio of 9:1 (quantitative ratio), and cells were determined by indirect competition ELISA. The supernatant was screened for positive wells. The positive wells were cloned by limiting dilution until a hybridoma cell line secreting the monoclonal antibody was obtained.
  • the mouse hybridoma cell line E-2-4 of dinitramine was screened.
  • the hybridoma cell line was deposited on May 21, 2012 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, Beijing, China) with the accession number CGMCC No. 6143.
  • the antibody secreted by the hybridoma cell line has good specificity for dinitramine, and the sensitivity can reach lg/L.
  • the affinity constant of the monoclonal antibody is determined by ELISA.
  • the dinitramine monoclonal hybridoma cell line ⁇ -2-4 was prepared into a cell suspension of 6 cells/mL with a cryopreservation solution, and stored for a long time in liquid nitrogen. When resuscitation, remove the cryotube and immediately put it into a 37 °C water bath for rapid fusion. After centrifugation to remove the frozen solution, transfer it to a culture flask for cultivation.
  • the concentrated washing solution diluted 20 times was washed twice, the liquid in the well was removed, and the residual water was absorbed by the absorbent paper, then 150-200 ⁇ blocking solution was added to each well, and the mixture was incubated at 37 ° C for 2 hours, and the liquid in the well was decanted. After drying, vacuum-sealed with aluminum film and stored at 4 °C in a dry place.
  • the coating solution was a carbonate buffer having a pH of 9.1-9.6 and 0.1-0.2 mol/L.
  • the blocking solution contained 5-10 wt% calf serum and 0.05 wt%.
  • the anti-antibody was coupled to horseradish peroxidase (HRP) using a modified sodium periodate method.
  • the traditional sodium periodate method requires a molar ratio of enzyme to anti-antibody in the reaction system of 4:1; since horseradish peroxidase produces many sites that bind to anti-antibodies under the action of strong oxidation, thus activated
  • the horseradish peroxidase molecule acts as a bridge connecting the molecules, reducing the enzymatic activity of the enzyme label, and mixing the prepared conjugate with many polymers. To solve this problem, we have improved the traditional method. , which is:
  • the blocking process of the amino group is omitted because the amino group capable of producing a self-amino group is practically few.
  • the horseradish peroxidase:antibody molar ratio was reduced to 2:1. Improved method is more traditional The method is simple and the loss of enzyme activity is reduced. 4. Using the above kit to detect residual dinitramine in the sample
  • a microplate reader purchased from Thermo Scientific, model MK3
  • the absorbance average value (B) of the obtained standard solution of each concentration was divided by the absorbance value (B Q ) of the first standard solution (0 standard) and multiplied by 100% to obtain a percent absorbance value.
  • Percent absorbance value (%) x ioo%
  • B is the average absorbance value of the standard solution or the sample solution
  • B Q is the average absorbance value of the standard solution of 0 g/L.
  • the concentration of the dinitramine standard (g/L) is the X-axis and the percent absorbance is the Y-axis.
  • the standard curve is drawn (Fig. 2).
  • the analysis of the detection results in the present invention can also be carried out by using the regression equation method to calculate the concentration of the sample solution.
  • the detection sensitivity of the kit for the preparation of the monoclonal antibody is determined by the precision, the accuracy, the detection limit and the stability index of the kit of the present invention.
  • the detection sensitivity of the kit for the dinitramine is O. lg / L, the accuracy is 80% - 100%, precision The degree is less than 15%.
  • Enzyme-linked immunosorbent assay can qualitatively and quantitatively detect trace residual harmful substances, and has low requirements on instruments, low cost, quick and easy operation, simple sample preparation process, low professional requirements for detectors, high sensitivity, and can be applied to a large number of sites. Rapid sample testing.
  • a batch of enzyme-labeled plates were taken from the enzyme-labeled plates prepared in three different time periods, and 10 kits were taken from each batch. 20 microwells were extracted from each plate, and the absorbance of the 9 g/L standard solution was determined. , calculate the coefficient of variation.
  • the precision is less than or equal to 25%.
  • the average recovery range of the concentration of 40 mg/kg and 80 g/kg dinitramine in the feed sample ranged from 86.9% to 92.5%
  • the intra-assay coefficient of variation ranged from 8.6 to 8%.
  • the inter-assay coefficient of variation is between 11.5% and 13.4%.
  • the average recovery of the two concentrations of dinitramine in 10 g/kg and 20 g/kg in chicken and chicken liver samples ranged from 80.1% to 89.7%, and the intra-assay coefficient of variation ranged from 9.6 to 12.3. Between %, the inter-assay coefficient of variation is between 10.5% and 14.5%.
  • a negative sample containing no dinitramine was taken and 20 times were tested with the kit. The average value of the results plus 3 standard deviations was used as the minimum detection limit of the kit.
  • the kit is stored at 2 to 8 ° C. After 12 months of measurement, the maximum absorbance of the kit (zero Standard), 50% inhibitory concentration, and actual measured values of dinitramine addition are within the normal range. It is considered that during the transportation and use, abnormal storage conditions will occur.
  • the kit is placed under the storage condition of 37 ° C for 6 days to carry out accelerated aging test. The results show that the indicators of the kit fully meet the requirements. Taking into account the freezing of the kit, the kit was frozen in a -20 ° C refrigerator for 5 days, and the results also showed that the kit parameters were completely normal. From the above results, it can be concluded that the kit can be stored at 2 to 8 ° C for more than 12 months.
  • Kit for the preparation of polyclonal antibodies Determination of the precision, accuracy, limit of detection and stability of the kits prepared with monoclonal antibodies, determined by the method described above
  • the polyclonal antibody kit has a precision of 6.9%-14.2%, an accuracy of 60%-100%, a kit sensitivity of 3.67 g/kg, and a kit that can hold 12 at 2 ⁇ 8 °C. More than a month.
  • the concentration is 0 g / L, 1 g / L, 3 g / L, 9 g / L, 27 g / L, 81 g / L;
  • the substrate color developing solution is composed of liquid A and liquid B.
  • the substrate coloring liquid A is urea peroxide or hydrogen peroxide
  • the substrate coloring liquid liquid B is tetramethylbenzidine or o-phenylenediamine.
  • the stop solution is 0.5-2 mol/L hydrochloric acid or sulfuric acid;
  • the concentrated washing solution is a 0.2-0.4 mol/L phosphate buffer solution having a pH of 7.1-7.6, containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide;
  • the concentrated solution is a phosphate buffer having a pH of 7.2-7.8 and 0.1-0.2 mol/L.
  • the substrate coloring solution is composed of liquid A and liquid B.
  • the substrate coloring liquid A is urea peroxide or hydrogen peroxide
  • the substrate coloring liquid B is tetramethylbenzidine or o-phenylenediamine.
  • the stop solution is 0.5-2 mol/L hydrochloric acid or sulfuric acid;
  • the concentrated washing solution is a 0.2-0.4 mol/L phosphate buffer solution having a pH of 7.1-7.6, containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide;
  • the concentrated solution is a phosphate buffer having a pH of 7.2-7.8 and 0.1-0.2 mol/L.
  • Example 4 A kit containing a dinitramine hapten and a carrier protein conjugate as a coating
  • Enzyme-labeled antibody working solution a monoclonal antibody of dinitramine labeled with horseradish peroxidase or a polyclonal antibody of dinitramine labeled with horseradish peroxidase;
  • the concentration is 0 g / L, 1 g / L, 3 g / L, 9 g / L, 27 g / L, 81 g / L;
  • the substrate color developing solution is composed of liquid A and liquid B.
  • the substrate coloring liquid A is urea peroxide or hydrogen peroxide
  • the substrate coloring liquid liquid B is tetramethylbenzidine or o-phenylenediamine.
  • the stop solution is 0.5-2 mol/L hydrochloric acid or sulfuric acid;
  • the concentrated washing solution is a 0.2-0.4 mol/L phosphate buffer solution having a pH of 7.1-7.6, containing 0.2-1.0 wt% Tween-20 and 0.01-0.06 wt% sodium azide;
  • the concentrated solution is a phosphate buffer having a pH of 7.2-7.8 and 0.1-0.2 mol/L.

Abstract

L'invention a trait à un kit ELISA de détection lié à l'enzyme dinitolmide et à son application. Ce kit ELISA lié à une enzyme comprend des anticorps spécifiques du dinitolmide, un péridium et des marqueurs enzymatiques, lesdits anticorps spécifiques du dinitolmide étant des anticorps monoclonaux de dinitolmide ou des anticorps polyclonaux de dinitolmide. Lorsque le péridium est un semi-antigène de dinitolmide conjugué avec une protéine-support, les marqueurs enzymatiques sont des anti-anticorps marqueurs enzymatiques, des anticorps monoclonaux ou polyclonaux anti-dinitolmide spécifiques de marqueurs enzymatiques. Lorsque le péridium est un anticorps spécifique du dinitolmide ou un anticorps anti-dinitolmide, les marqueurs enzymatiques sont des semi-antigènes de dinitolmide de marqueurs enzymatiques.
PCT/CN2012/078164 2012-07-04 2012-07-04 Kit elisa de détection lié à l'enzyme dinitolmide et son application WO2014005298A1 (fr)

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PCT/CN2012/078164 WO2014005298A1 (fr) 2012-07-04 2012-07-04 Kit elisa de détection lié à l'enzyme dinitolmide et son application
US14/412,317 US10234470B2 (en) 2012-07-04 2012-07-04 Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof

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PCT/CN2012/078164 WO2014005298A1 (fr) 2012-07-04 2012-07-04 Kit elisa de détection lié à l'enzyme dinitolmide et son application

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CN113092392A (zh) * 2021-03-05 2021-07-09 苏州西山中科药物研究开发有限公司 一种检测猴血清中靶向可溶性蛋白的单克隆抗体药物总浓度的通用型方法

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CN111812316B (zh) * 2020-06-03 2022-11-18 北京勤邦生物技术有限公司 一种甲氰菊酯人工抗原在酶联免疫试剂盒中的应用
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CN113092392A (zh) * 2021-03-05 2021-07-09 苏州西山中科药物研究开发有限公司 一种检测猴血清中靶向可溶性蛋白的单克隆抗体药物总浓度的通用型方法
CN113092392B (zh) * 2021-03-05 2022-11-08 苏州西山中科药物研究开发有限公司 一种检测猴血清中靶向可溶性蛋白的单克隆抗体药物总浓度的通用型方法
CN113049832A (zh) * 2021-03-15 2021-06-29 上海交通大学 定量检测牛乳中过敏原α-乳白蛋白的双抗体夹心法

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